The triad junction of skeletal muscle is comprised of a single invagination of the plasma membrane that plunges into the cytoplasm (the transverse-tubules or T-tubules) that is juxtaposed with two sections of the terminal cisternae of the sarcoplasmic reticulum (SR). Screening of an antibody library for novel proteins that localize to the triad junction by immunostaining identified proteins that are implicated in excitation-contraction (E-C) coupling and other aspects of Ca2+ handling in skeletal muscle. One protein identified during the screening of this library was a novel transmembrane protein called, mitsugumin29 (MG29).
MG29 is nearly exclusively expressed in skeletal muscle fibers, although some minor levels of expression can be resolved in the kidney, and contains four transmembrane domains that allow the protein to localize at both the transverse (T-) tubular membrane and SR membranes of the triad junction. This subcellular distribution suggest MG29 may mediate communication between the T-tubular and junctional SR membrane. The protein structure of MG29 is homologous in amino acid sequence and shares characteristic structural features with the members of the synaptophysin family of transmembrane proteins essential for neurotransmitter release.
Synaptophysin was originally identified as an abundant and highly immunogenic membrane protein of small synaptic vesicles that is also found in dense-core chromaffin and neurosecretory granules. Synaptophysin and its homologues, synaptoporin (or synaptophysin II) and pantophysin, share a common transmembrane organization, with four membrane-spanning regions and cytoplasmic amino and carboxy termini.
A unique feature of synaptophysin is that it has an oligomeric structure, leading to the proposal that synaptophysin may be a component of the fusion pore that forms during neurotransmitter release. Moreover, Alder et al. have shown that antisense oligonucleotides complementary to the synaptophysin mRNA reduce Ca2+-dependent glutamate secretion from Xenopus oocytes induced by injection of total brain mRNA. Microinjection of synaptophysin antibody into motor neurons blocked neuromuscular transmission. These data are consistent with synaptophysin being essential for neurotransmitter secretion. However, genetic approaches to identify the function of synaptophysin have not been successful; mutant mice lacking synaptophysin show a normal phenotype. This may reflect compensation by synaptoporin or other synaptophysin family members. Indeed, mice doubly deficient in synaptophysin and synaptogyrin display defects in synaptic plasticity.
Synaptophysin has been proposed to play a structural role in vesicle formation. Based on its high capacity to bind cholesterol, synaptophysin has been implicated in the generation of membrane curvature during synaptic vesicle biogenesis. Synaptophysin is also known to tightly interact with other proteins of the synaptic vesicle membrane, i.e. synaptobrevin and the vacuolar H+-ATPase. These interactions are thought to regulate exocytotic membrane fusion at the level of the SNARE complex or fusion pore formation. The latter idea is supported by studies on yeast vacuole fusion that implicate the vacuolar ATPase directly participate in membrane fusion.
The similarities between MG29 and synaptophysin prompted an investigation into whether MG29 plays an important role in modulation of membrane structures in skeletal muscle. Skeletal muscles are among the most plastic tissue in nature, and normal muscle physiology requires the formation and maintenance of the complex membrane structures. Throughout development, aging and other processes including fatigue require constant adaptations of the skeletal muscle system, thus identification and characterization of genes and proteins involved with plasticity in skeletal muscle membrane structures is essential to understand muscle physiology, as well as treating and diagnosing pathologies related to muscle dysfunction, including diabetes.
Skeletal muscle is a form of striated muscle tissue existing under control of the somatic nervous system. It is one of three major muscle types, the others being cardiac and smooth muscle. Skeletal muscle accounts for as much as 80% of insulin-sensitive glucose metabolism and a substantial portion of whole body glucose metabolism in humans. Insulin resistance (IR) of skeletal muscle is a major risk factor for metabolic disease and is characteristic of type 2 diabetes mellitus, obesity, and hypertension. A substantial body of literature identifies the proximal steps of glucose metabolism, those of glucose delivery, transport, and phosphorylation, as key loci of insulin action in health and as determinants of skeletal muscle insulin resistance.
Therefore, an understanding of the mechanism and regulation of muscle glucose utilization is necessary to determine the nature of the defects present in metabolic diseases such as obesity and insulin-resistant diabetes. Glucose transport, the initial step in glucose utilization, is often considered rate determining in glucose utilization in muscle. One approach to assessing the balance of transport and phosphorylation in determining glucose utilization is to establish how these processes are coupled at the cellular level
Diabetes mellitus is a metabolic disorder characterized by an inability to control blood glucose levels due to either a loss of the insulin-producing beta cells of the islets of Langerhans in the pancrease, which leads to insulin deficiency (i.e., Type I diabetes), and/or due to insulin resistance (i.e., Type II diabetes or NIDD).
Type I diabetes can be further classified as immune-mediated or idiopathic. The majority of type 1 diabetes is of the immune-mediated nature, where beta cell loss is a T-cell mediated autoimmune attack. There is no known preventive measure against type 1 diabetes, which causes approximately 10% of diabetes mellitus cases in North America and Europe. Most affected people are otherwise healthy and of a healthy weight when onset occurs. Sensitivity and responsiveness to insulin are usually normal, especially in the early stages. Type 1 diabetes can affect children or adults but was traditionally termed “juvenile diabetes” because it represents a majority of the diabetes cases in children.
With Type II diabetes the defective responsiveness of body tissues to insulin is believed to involve the insulin receptor. However, the specific defects are not known. In the early stage of Type II diabetes, the predominant abnormality is reduced insulin sensitivity. At this stage hyperglycemia can generally be reversed by a variety of measures and medications that improve insulin sensitivity or reduce glucose production by the liver. As the disease progresses, the impairment of insulin secretion occurs, and therapeutic replacement of insulin may sometimes become necessary in certain patients.
If the amount of insulin available is insufficient, if cells respond poorly to the effects of insulin (insulin insensitivity or resistance), or if the insulin itself is defective, then glucose will not have its usual effect so that glucose will not be absorbed properly by those body cells that require it nor will it be stored appropriately in the liver and muscles. The net effect is persistent high levels of blood glucose, poor protein synthesis, and other metabolic derangements, such as acidosis.
When the glucose concentration in the blood is raised beyond its renal threshold (about 10 mmol/L, although this may be altered in certain conditions, such as pregnancy), reabsorption of glucose in the proximal renal tubuli is incomplete, and part of the glucose remains in the urine (glycosuria). This increases the osmotic pressure of the urine and inhibits reabsorption of water by the kidney, resulting in increased urine production (polyuria) and increased fluid loss. Lost blood volume will be replaced osmotically from water held in body cells and other body compartments, causing dehydration and increased thirst.
Lack of adequate treatment of diabetes can lead to acute complications, including hypoglycemia, diabetic ketoacidosis, nonketonic hyperglycemia, or nonketotic hyperosmolar coma. Serious long-term complications include cardiovascular disease, e.g., heart attack, stroke, arterial diseases (e.g., coronary artery disease); neuropathy; renal failure; retinal damage; erectile dysfunction; blindness, slow healing wounds; and amputation.
As of 2000 at least 171 million people worldwide suffer from diabetes, or 2.8% of the population. Type 2 diabetes is by far the most common, affecting 90 to 95% of the U.S. diabetes population (Wild S, Roglic G, Green A, Sicree R, King H (May 2004). “Global prevalence of diabetes: estimates for 2000 and projections for 2030”. Diabetes Care 27 (5): 1047-53). Accordingly, there exists an ongoing need for the development of pharmaceutical modulators of muscle function for the treatment of conditions related to muscle dysfunction, including diabetes.